The field of the invention relates to the regulation of circadian rhythms.
Circadian rhythms in mammals are regulated by a master clock located in the suprachiasmatic nucleus (SCN) of the brain (Klein et al., Suprachiasmatic nucleus: The Mind""s Clock, Oxford University Press, New York, 1991; Reppert and Weaver, Cell 89:487-490, 1997). Environmental light-dark cycles entrain the SCN clock to the 24-hr day via direct and indirect retinal projections. The timekeeping capability of the SCN is expressed at the level of single neurons (Welsh et al., Neuron 14:697-706, 1995).
The SCN clock mechanism is cell-autonomous, possibly based on transcriptional and translational negative feedback loops (Reppert, Neuron 21:1-4, 1998). Precedent for such a mechanism has been described for circadian clocks in the fly Drosophila melanogaster. 
In the fly, autoregulatory transcriptional loops occur in which protein products of clock genes periodically enter the nucleus to suppress their own transcription. This feedback loop involves dynamic regulation of the clock genes period (per) and timeless (Tim). As the levels of PER and TIM rise, they are phosphorylated, form heterodimers, and are then translocated to the nucleus where they negatively regulate their own transcription (Saez and Young, Neuron 17:1-920, 1996; Darlington et al., Science 280:1599-1603, 1998). Negative transcriptional regulation appears to involve interference with drosophila CLOCK:drosophila dBMAL-1 (dCLOCK:dBMAL-1) and may be mediated by direct interaction of PER and TIM with dCLOCK. dCLOCK and dBMAL-1 are positive factors which drive Per and Tim transcriptional activation by binding to CACGTG E-box enhancers in the promoters of Per and Tim (Allada et al., Cell 93:791-804, 1998; Rutila et al., Cell 93:805-814, 1998; Darlington et al., supra; Hao et al., Mol. Cell Biol. 17:3687-3693, 1997). The temporal phosphorylation of PER provides at least part of the time delay between transcription and PER-TIM negative feedback necessary to sustain a 24-hr molecular oscillation in drosophila (Price et al., Cell 94:83-95, 1998).
The invention is based, in part, on the discovery that the core clockwork in the SCN is comprised of interacting feedback loops. It was discovered that cryptochrome (CRY) proteins are critical players in the negative limb of the mammalian clock feedback loop and Period 2 (PER2) protein is a critical regulator of the Bmal-1 loop. The CRY proteins and PER2 protein therefore function as important modulators of mammalian circadian rhythm.
It was discovered that mammalian CRY proteins can translocate from the cytoplasm to the nucleus of a cell and inhibit CLOCK:BMAL-1 induced transcription. It was also discovered that CRY proteins can homodimerize or heterodimerize with other circadian proteins. The ability of CRY to heterodimerize with other proteins provides a mechanism whereby CRY can modulate the activity of other circadian proteins. For example, mouse CRY proteins can function as dimeric and potentially trimeric partners for mouse PER proteins; these interactions lead to the nuclear translocation of PER. Once in the nucleus, PER can inhibit CLOCK:BMAL-1 induced transcription. In addition, it was discovered that mouse CRY can form heterodimeric complexes with mouse TIM. The interaction of TIM with CRY may have a role in modulating the negative feedback of mouse PER and/or mouse CRY rhythms. Thus, the compounds which can disrupt the interaction of CRY with itself and other circadian proteins can be used to reset the circadian clock.
In addition, it was discovered that PER2 positively regulates transcription of the Bmal-1 gene. The ability of PER2 to positively regulate the transcription of Bmal-1 indicates that PER2 controls the rhythmic regulation of Bmal-1. The availability of BMAL-1 is critical for restarting the circadian clock loop. When BMAL-1 is available, it heterodimerizes with CLOCK, thereby driving the transcription of Per genes (e.g., in the mouse(m), mPER1-3) and Cryptochrome genes (e.g., mouse mCry1 and mCry2). Compounds which can disrupt the ability of PER2 to positively activate Bmal-1, or compounds which can modulate transcription of Bmal-1, can be used to reset the circadian clock.
Accordingly, the invention includes a method for identifying a compound which binds to a mammalian CRY protein. The method, which is useful as a quick initial screen for CRY agonists and antagonists, includes contacting the CRY protein with a test compound and determining whether the latter binds to the CRY protein. Binding by the test compound to the CRY protein indicates that the test compound is a CRY protein binding compound. For ease of detection, the test compound can be labeled, e.g., radiolabeled. The CRY protein can any mammalian CRY protein such as a CRY from a mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep, pig, non-human, primate, or human. In particular, the CRY protein is a mouse CRY1 or CRY2 or human CRY1 or CRY2.
The method may further include contacting the test compound with: a CRY protein in the presence of a PER protein; a CRY protein in the presence of a TIM protein; a CRY protein in the presence of a CLOCK:BMAL-1 complex; or a CRY protein in the presence of a BMAL-1 protein; and determining whether the test compound disrupts the association of the CRY protein with the PER, TIM, CLOCK:BMAL-1, or BMAL-1 protein, as the case may be; wherein a decrease in the association in the presence of the test compound compared to the association in the absence of the test compound indicates that the test compound disrupts the association of the CRY protein with the indicated binding partner. The PER protein can any mammalian PER protein such as mouse, rat, rabbit, goat, horse, cow, pig, dog, cat or human. For example, the PER protein may be mouse or human PER1, PER2 or PER3.
The method can further include contacting the test compound with the first CRY protein in the presence of a second CRY protein and determining whether the test compound disrupts the association of the first CRY protein with the second CRY protein, wherein the second CRY protein has an amino acid sequence the same as or different than the first CRY protein, and wherein a decrease in the association in the presence of the test compound compared to the association in the absence of the test compound indicates that the test compound disrupts the association of the first CRY protein and the second CRY protein. The first and second CRY proteins can be any mammalian CRY protein such as a CRY from a mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep, non-human, primate or human. For example, each CRY protein can be a mouse or human CRY1 or CRY2 and the second CRY protein is a mouse CRY1 or CRY2.
The method can further include providing a cell or cell-free system which includes a CRY protein, a CLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively linked to a reporter gene. The method includes introducing the test compound into the cell or cell-free system and assaying for transcription of the reporter gene, wherein an increase in transcription in the presence of the compound compared to transcription in the absence of the compound indicates that the compound blocks CRY-induced inhibition of CLOCK:BMAL-1-mediated transcription in a cell. The cell can be any cell type, such as a cultured mammalian cell, e.g., a NIH3T3 cell, a COS7 cell, or a clock neuron. The reporter gene can be a gene that encodes a detectable marker, e.g., luciferase.
The invention further includes a method for identifying a compound which disrupts the association of a CRY protein and a second protein or protein complex, which can be any of the following: a PER protein, a TIM protein, a BMAL-1 protein, a second CRY protein, or a CLOCK:BMAL-1 complex. The method includes contacting a test compound with the CRY protein in the presence of the second protein (or protein complex) and determining whether the test compound disrupts the association of the CRY protein and the second protein (or protein complex), wherein a decrease in the association in the presence of the test compound compared to the association in the absence of the test compound indicates that the test compound disrupts the association of the CRY protein and the second protein. The first and second CRY proteins can be any mammalian CRY protein such as a CRY protein from a mouse, rat, rabbit, goat, horse, cow, sheep, pig, dog, cat, non-human primate or human, e.g., a mouse or human CRY1 or CRY2. The PER protein can be any mammalian PER protein as described above, e.g., a mouse PER1, PER2 or PER3. The TIM protein can be any mammalian TIM protein as described above, e.g., a mouse or human TIM protein. The CLOCK and the BMAL-1 proteins can be any mammalian CLOCK and BMAL-1 proteins as described above, particularly mouse or human.
Also within the invention is a method for identifying a compound that blocks CRY-induced inhibition of CLOCK:BMAL-1 transcription in a cell. The method includes providing a cell comprising a CRY protein, a CLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively linked to a reporter gene; introducing the compound into the cell or a cell-free transcription system; and assaying for transcription of the reporter gene, wherein an increase in transcription in the presence of the compound compared to transcription in the absence of the compound indicates that the compound blocks CRY-induced inhibition of CLOCK:BMAL-1-mediated transcription. The cell can be any cell type, such as a cultured mammalian cell, e.g., a NIH3T3 cell, a COS7 cell or a clock neuron. The reporter gene can be gene that encodes a detectable marker, e.g., luciferase.
The invention further includes a method for identifying a compound that activates or inhibits the transcription of Per2. The method includes providing a cell including a mammalian Per2 regulatory sequence operatively linked to a reporter gene, introducing a test compound into the cell, and assaying for transcription of the reporter gene in the cell. A decrease in transcription in the presence of the compound compared to transcription in the absence of the compound indicates that the compound inhibits Per2 transcription in a cell. Likewise, an increase of transcription in the presence of the compound compared to transcription in the absence of the compound indicates that the compound inhibits Per2 transcription in a cell. The cell can be any cell that can generate circadian rhythms, such as a NIH3T3 cell, a Cos-7 cell or a clock neuron. The reporter gene can be any detectable marker, e.g., a luciferase, a chloramphenicol acetyl transferase, a beta- galactosidase, an alkaline phosphate, or a fluorescent protein such as green fluorescent protein. The Per2 regulatory sequence can be any mammalian Per2 regulatory sequence, e.g., from a mouse, a rat, a rabbit, a goat, a horse, a cow, a pig, a dog, a cat, a sheep, a non-human primate, or a human. In particular, the Per2 regulatory sequence can be a mouse Per2 regulatory sequence (SEQ ID NO:3).
Also within the invention is a method of determining if a candidate compound positively regulates the expression of Bmal-1. The method includes providing a transgenic animal whose somatic and germ cells comprise a disrupted Per2 gene, the disruption being sufficient to inhibit the ability of Per2 to positively regulate Bmal-1, administering a test compound to the mouse, and detecting Bmal-1 expression, wherein an increase in the expression of Bmal-1 indicates that the compound can positively regulate expression of Bmal-1.
The invention also features a method of modulating circadian-clock controlled rhythms in a cell including comprising introducing into a cell an expression vector encoding a BMAL-1 protein such that an effective amount of the BMAL-1 protein is produced in the cell, thereby modulating circadian-clock controlled rhythms. The BMAL-1 can be any mammalian BMAL-1, e.g., that of a mouse, a rat, a rabbit, a goat, a horse, a cow, a dog, a cat, a sheep, a non-human primate, or a human BMAL-1.
Also within the invention is a method of modulating circadian-clock controlled rhythms in a cell comprising introducing into the cell an effective amount of an oligonucleotide antisense to a part, or all, of a mammalian Bmal-1, thereby inhibiting expression of Bmal-1 in the cell and modulating circadian-clock rhythms. Oligonucleotides can be antisense to any mammalian Bmal-1, e.g., Bmal-1 from a mouse, a rat, a rabbit, a goat, a horse, a cow, a sheep, a non-human primate, or a human.
The invention further includes isolated nucleic acid molecules which are at least about 60% (or 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of mouse TIMELESS (TIM) (SEQ ID NO:1). The invention also features isolated nucleic acid molecules which include a fragment of at least 100 (e.g., at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 3745) nucleotides of the nucleotide sequence of SEQ ID NO: 1, or a complement thereof. The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least about 60% (or 70%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1, or a complement thereof
Also within the invention is an isolated polypeptide having an amino acid sequence that is at least about 60%, preferably 70%, 75%, 85%, 95%, or 98%, identical to the amino acid sequence of SEQ ID NO:2. Also within the invention are isolated polypeptides encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to the complement of SEQ ID NO: 1.
The invention also features isolated nucleic acid molecules which are at least about 60% (or 65%, 75%, 85%, 95%, or 98%) identical to the mouse Per2 upstream sequence (SEQ ID NO:3) containing a sequence controlling expression of mouse Per2. The invention also features isolated nucleic acid molecules which include a fragment of at least 100 (e.g., at least 200, 300, 400, 500, 600, 700, 800, 900, or 950) nucleotides of the nucleotide sequence of SEQ ID NO:3, or a complement thereof.
The invention also includes nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. The nucleic acid molecules can be, for example, at least 20 (e.g. at least about 30, 40, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 3745) nucleotides in length.
Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule described herein. The vector or nucleic acid molecule can be provided in a host cell. Such cells may be utilized for producing a polypeptide of the invention by culturing the cells in a suitable medium.
Also within the invention are a substantially pure preparation of a mouse or human TIM, a mouse or human CRY:PER heterodimer, a CRY:TIM heterodimer, and a mammalian CRY:CRY homodimer.
Isolated antibodies, which specifically bind to mouse CRY, mouse PER, mouse TIM, mouse BMAL-1 are also within the invention.
As used herein, xe2x80x9cisolated DNAxe2x80x9d means either DNA with a non-naturally occurring sequence or DNA free of the genes that flank the DNA in the genome of the organism in which the DNA naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment.
As used herein, an regulatory sequence which is xe2x80x9coperably linkedxe2x80x9d to a second sequence (or vise versa) means that both are incorporated into a genetic construct so that the regulatory sequence effectively controls expression of a second sequence.
As used herein, a xe2x80x9csubstantially purexe2x80x9d protein refers to a protein which either (Klein et al., (1991). Suprachiasmatic nucleus: The Mind""s Clock, Oxford University Press, New York. has a non-naturally occurring sequence (e.g., mutated, truncated, chimeric, or completely artificial), or (D. R. Weaver, J. Biol. Rhythms 13, 100 (1998) has a naturally occurring sequence but is not accompanied by or at least partially separated from, components that naturally accompany it. Typically, the protein is substantially pure when it is at least 60% (by weight) free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure protein can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the protein or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically synthesized protein or a recombinant protein produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure proteins include those having sequences derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
As used herein, the term xe2x80x9cvectorxe2x80x9d refers to a replicable nucleic acid construct. Examples of vectors include plasmids and viral nucleic acids.
As used herein, a xe2x80x9ccircadian proteinxe2x80x9d refers to a protein that participates in the circadian timing system and controls circadian rhythm. Examples of circadian proteins include PER, TIM, CLOCK, and BMAL-1.
As used herein, an antibody that xe2x80x9cspecifically bindsxe2x80x9d a mouse or human CRY, PER or TIM, respectively, is an antibody that binds only to mouse or human CRY, PER or TIM and does not bind to (i) other molecules in a biological sample or (ii) CRY, PER or TIM of another organism.
As used herein, a xe2x80x9ctherapeutically effective amountxe2x80x9d is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal.
As used herein, a xe2x80x9creporter genexe2x80x9d means a gene whose expression can be assayed.
As used herein, the terms xe2x80x9cheterologous DNAxe2x80x9d or xe2x80x9cheterologous nucleic acidxe2x80x9d is meant to include DNA that does not occur naturally as part of the genome in which it is present, or DNA which is found in a location or locations in the genome that differs from that in which it occurs in nature, or occurs extra-chromasomally, e.g., as part of a plasmid.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will control. Unless otherwise indicated, materials, methods, and examples described herein are illustrative only and not intended to be limiting.
Various features and advantages of the invention will be apparent from the following detailed description and from the claims.